Lipid-polymer conjugates, their preparation and uses thereof

ABSTRACT

This invention provides low molecular weight lipid-GAG and phospholipids-GAG conjugates and methods of use thereof in suppressing, inhibiting, preventing, or treating a pathogenic effect on a cell, including, inter alia, infection with intracellular pathogens.

FIELD OF THE INVENTION

This invention provides low molecular weight lipid-GAG conjugates andmethods of use thereof in suppressing, inhibiting, preventing, ortreating a pathogenic effect on a cell, including, inter alia, infectionwith intracellular pathogens.

BACKGROUND OF THE INVENTION

Lipid-conjugates having a pharmacological activity of inhibiting theenzyme phospholipase A2 (PLA2, EC 3.1.1.4) are known in the prior art.Phospholipase A2 catalyzes the breakdown of phospholipids at the sn-2position to produce a fatty acid and a lysophospholipid. The activity ofthis enzyme has been correlated with various cell functions,particularly with the production of lipid mediators such as eicosanoidproduction (prostaglandins, thromboxanes and leukotrienes), plateletactivating factor and lysophospholipids. Lipid-conjugates may offer awider scope of protection of cells and organisms from injurious agentsand pathogenic processes, including the prevention and treatment ofmicrobial infections. Lipid-conjugates may offer a wider scope ofprotection of cells and organisms from injurious agents and pathogenicprocesses, including the prevention and treatment of microbialinfections.

Lipid-conjugates have been subjected to intensive laboratoryinvestigation in order to obtain a wider scope of protection of cellsand organisms from injurious agents, pathogenic and inflammatoryprocesses.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a lipid-polymerconjugate comprising a glycosaminoglycan (GAG) conjugated to aphospholipid (PL) wherein said conjugate is prepared by reacting saidGAG with said PL in a mass_(PL) to mass_(GAG) ratio from about 0.25:15to about 5:15, respectively.

In one embodiment, the present invention provides a lipid-polymerconjugate comprising a glycosaminglycan (GAG) conjugated to aphospholipid (PL) via an amide or ester linkage wherein the molecularweight of said GAG is between 5 to 20 kD.

In one embodiment, the present invention provides a lipid-polymerconjugate represented by the structure of the general formula (A):

-   -   wherein    -   L is a lipid or a phospholipid;    -   Z is either nothing, ethanolamine, serine, inositol, choline,        phosphate, or glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between L, Z, Y and X is either an amide or an        esteric bond;    -   wherein the molecular weight of said glycosaminoglycan is        between 5 kD and 20 kD.

In one embodiment, the present invention provides a process forpreparing a compound represented by the structure of the general formula(I):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein if Y is nothing the phosphatidylethanolamine is directly        linked to X via an amide bond and if Y is a spacer, said spacer        is directly linked to X via an amide or an esteric bond and to        said phosphatidylethanolamine via an amide bond;    -   comprising the steps of:    -   b. reacting a phospholipid (PL) with a glycosaminoglycan (GAG)        and a coupling agent, wherein the mass_(PL) to mass_(GAG) ratio        from about 0.25:15 to about 5:15, respectively;    -   c. filtering the reaction mixture from (a) to generate a        filtrate; and    -   d. extracting a product from a filtrate.

In one embodiment, the present invention provides a method of treatinginflammatory disorders in a subject, said method comprisingadministering to a subject suffering from an inflammatory disorder acomposition comprising a lipid-polymer conjugate comprising aglycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein saidconjugate is prepared by reacting said GAG with said PL in a mass_(PL)to mass_(GAG) ratio from about 0.25:15 to about 5:15, respectively. Inone embodiment, the present invention provides a method for decreasingexpression of proinflammatory chemokines, cytokines, or a combinationthereof comprising the step of administering a compound represented bythe structure of the general formula (A):

-   -   wherein    -   L is a lipid or a phospholipid;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between L, Z, Y and X is either an amide or an        esteric bond to a subject with high levels of proinflammatory        chemokines, cytokines, or a combination thereof.

In one embodiment, the present invention provides a method of activatingNF-κB, IL-6, IL-8, or a combination thereof in human airway epithelialcell lines comprising the step of administering to a subject a compoundrepresented by the structure of the general formula (A):

-   -   wherein    -   L is a lipid or a phospholipid;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between L, Z, Y and X is either an amide or an        esteric bond.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter regarded as the invention is particularly pointed outand distinctly claimed in the concluding portion of the specification.The invention, however, both as to organization and method of operation,together with objects, features, and advantages thereof, may best beunderstood by reference to the following detailed description when readwith the accompanying drawings in which:

FIG. 1 depicts a conceptual diagram of the reaction vessel featuresrequired to practice the methods of this invention.

FIG. 2 depicts an NMR spectrum of a hyaluronicacid-phosphatidylethanolamine conjugate (HyPE) prepared according toExample 5.

FIG. 3 is an HPLC chromatogram of HyPE prepared according to Example 5.

FIG. 4 depicts a schematic representation of the in vitro stimulation ofRAW 264.7 cells.

FIG. 5 depicts the mean XTT reduction (OD₄₅₀) by RAW 264.7 cells in theabsence of LPS. Error bars represent standard deviations.

FIG. 6 depicts the mean XTT reduction (OD₄₅₀) by LPS-stimulated RAW264.7 cells. Error bars represent standard deviations.

FIG. 7 depicts the mean TNF-α release from RAW 264.7 cells in theabsence of LPS. Error bars represent standard deviations.

FIG. 8 depicts the mean TNF-α release from LPS-stimulated RAW 264.7cells. Error bars represent standard deviations.

FIG. 9 depicts the mean IL-6 release from RAW 264.7 cells in the absenceof LPS. Error bars represent standard deviations.

FIG. 10 depicts the mean IL-6 release from LPS-stimulated RAW 264.7cells. Error bars represent standard deviations.

FIG. 11 depicts the mean IP-10 release from RAW 264.7 cells in theabsence of LPS. Error bars represent standard deviations.

FIG. 12 depicts the mean IP-10 release from LPS-stimulated RAW 264.7cells. Error bars represent standard deviations.

FIG. 13 depicts the mean PGE₂ release from RAW 264.7 cells in theabsence of LPS. Error bars represent standard deviations.

FIG. 14 depicts the mean PGE₂ release from LPS-stimulated RAW 264.7cells. Error bars represent standard deviations.

FIG. 15 depicts dose-response curves for TNF-α production (+LPS). Datafit using Prism 4, Sigmoidal dose-response curve (variable slope):Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log ofTest Article concentration, Y is the response. Constraints Bottom=0,Top=100.

FIG. 16 depicts dose-response curves for IL-6 production (+LPS). Datafit using Prism 4, Sigmoidal dose-response curve (variable slope):Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log ofTest Article concentration, Y is the response. Constraints Bottom=0,Top=100.

FIG. 17 depicts dose-response curves for IP-10 production (+LPS). Datafit using Prism 4, Sigmoidal dose-response curve (variable slope):Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log ofTest Article concentration, Y is the response. Constraints Bottom=0,Top=100.

FIG. 18 depicts dose-response curves for PGE₂ production (+LPS). Datafit using Prism 4, Sigmoidal dose-response curve (variable slope):Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log ofTest Article concentration, Y is the response. Constraints Bottom=0,Top=100.

FIG. 19 is the chromatogram from the SEC-MALS molecular weight analysisof low molecular weight sodium hyaluronate. The red line pertains to thelight scattering signal. The blue line refers to the refractive indexsignal.

FIG. 20 is the SEC-MALS determined distribution of molecular weight oflow molecular weight sodium hyaluronate.

FIG. 21 is the UV spectrum of sample 208-088 (low molecular weightsodium hyaluronate).

FIG. 22 depicts the mean XTT reduction (OD₄₅₀) by RAW 264.7 cells in theabsence of LPS. Error bars represent standard deviations.

FIG. 23 depicts the mean XTT reduction (OD₄₅₀) by LPS-stimulated RAW264.7 cells. Error bars represent standard deviations.

FIG. 24 depicts the mean TNF-α release from RAW 264.7 cells in theabsence of LPS. Error bars represent standard deviations.

FIG. 25 depicts the mean TNF-α release from LPS-stimulated RAW 264.7cells. Error bars represent standard deviations.

FIG. 26 depicts the mean IL-6 release from RAW 264.7 cells in theabsence of LPS. Error bars represent standard deviations.

FIG. 27 depicts the mean IL-6 release from LPS-stimulated RAW 264.7cells. Error bars represent standard deviations.

FIG. 28 depicts the mean IP-10 release from RAW 264.7 cells in theabsence of LPS. Error bars represent standard deviations.

FIG. 29 depicts the mean IP-10 release from LPS-stimulated RAW 264.7cells. Error bars represent standard deviations.

FIG. 30 depicts the mean PGE₂ release from RAW 264.7 cells in theabsence of LPS. Error bars represent standard deviations.

FIG. 31 depicts the mean PGE₂ release from LPS-stimulated RAW 264.7cells. Error bars represent standard deviations.

FIG. 32 depicts dose-response curves for TNF-α production (+LPS). Datafit using Prism 4, Sigmoidal dose-response curve (variable slope):Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log ofTest Article concentration, Y is the response. Constraints Bottom=0,Top=100.

FIG. 33 depicts dose-response curves for IL-6 production (+LPS). Datafit using Prism 4, Sigmoidal dose-response curve (variable slope):Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log ofTest Article concentration, Y is the response. Constraints Bottom=0,Top=100.

FIG. 34 depicts dose-response curves for IP-10 production (+LPS). Datafit using Prism 4, Sigmoidal dose-response curve (variable slope):Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log ofTest Article concentration, Y is the response. Constraints Bottom=0,Top=100.

FIG. 35 depicts dose-response curves for PGE₂ production (+LPS). Datafit using Prism 4, Sigmoidal dose-response curve (variable slope):Y=Bottom+(Top+Bottom)/(1+10̂((LOGIC50−X)*HillSlope)). X is the log ofTest Article concentration, Y is the response. Constraints Bottom=0,Top=100.

FIG. 36 depicts a photograph of the actual reaction vessel used for thepreparation of HyPE. The chiller is behind the reaction vessel and thedoor on the sound-proof container is open to reveal the ultrasoundflow-cell.

FIG. 37 depicts a chromatogram of the HyPE reaction from Example 11after 2 hours.

FIG. 38 depicts a chromatogram of the HyPE reaction from Example 11after 6 hours.

FIG. 39 depicts the GPC analysis of final HyPE isolated from Example 11.

FIG. 40 depicts the NMR spectrum of final HyPE isolated from Example 11and treated with 1 drop of 4% NaOD.

It will be appreciated that for simplicity and clarity of illustration,elements shown in the figures have not necessarily been drawn to scale.For example, the dimensions of some of the elements may be exaggeratedrelative to other elements for clarity. Further, where consideredappropriate, reference numerals may be repeated among the figures toindicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the invention.However, it will be understood by those skilled in the art that thepresent invention may be practiced without these specific details. Inother instances, well-known methods, procedures, and components have notbeen described in detail so as not to obscure the present invention.

Abbreviations used to specify chemicals and reagents used in theprocesses described herein are readily recognized by one skilled in theart. For the purposes of this invention, it will be understood that DCCrefers to dicyclohexylcarbodiimide, EDAC refers to1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), BOP refersto Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate, PyBOP refers tobenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, HATUrefers to O-(7-Azabenzotriazole-1-yl)-N,N,N′N′-tetramethyluroniumhexafluorophosphate, TSTU refers toO—(N-Succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, HOBTrefers to hydroxybenzotriazole and HOAT refers to1-hydroxy-7-aza-benzotriazole.

Herein, the term “lipid” refers to all types of lipids includingphospholipids, glycerolipids, sphingolipids, sterol lipids, prenollipids, saccharolipids and the like.

This invention provides, in one embodiment, a lipid-polymer conjugatewhich is useful in some embodiments for the treatment of inflammatorydisorders.

In some embodiments, this invention provides a method for thepreparation of the lipid-polymer conjugates of this invention. In someembodiments, this invention provides a method for the use of thelipid-polymer conjugates of this invention.

In one embodiment, this invention provides a lipid-polymer conjugatecomprising a glycosaminoglycan (GAG) conjugated to a phospholipid (PL)wherein said conjugate is prepared by reacting said GAG with said PL ina mass_(PL) to mass_(GAG) ratio from about 0.25:15 to about 5:15,respectively.

In another embodiment, said mass_(PL) to mass_(GAG) ratio is about0.25:15. In another embodiment, said mass_(PL) to mass_(GAG) ratio isabout 0.5:15. In another embodiment, said mass_(PL) to mass_(GAG) ratiois about 1:15. In another embodiment, said mass_(PL) to mass_(GAG) ratiois about 2:15. In another embodiment, said mass_(PL) to mass_(GAG) ratiois about 5:15.

In one embodiment, the present invention provides a lipid-polymerconjugate comprising a glycosaminglycan (GAG) conjugated to aphospholipid (PL) via an amide or ester linkage wherein the molecularweight of said GAG is between 5 to 20 kD.

In another embodiment, said GAG of the lipid-conjugate compound of thisinvention is hyaluronic acid, heparin, heparan sulfate, chondroitin,chondroitin sulfate, dermatan sulfate or keratan sulfate. In anotherembodiment, said GAG is hyaluronic acid. In another embodiment, said GAGis heparin. In another embodiment, said GAG is chondroitin. In anotherembodiment, said GAG is chondroitin sulfate. In another embodiment, saidGAG is dermatan sulfate, in another embodiment, said GAG is keratansulfate.

In another embodiment, said chondroitin sulfate ischondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof. Inanother embodiment, said dermatan sulfate is dermatan-6-sulfate,dermatan-4-sulfate or a derivative thereof.

In another embodiment, said PL of the lipid-conjugate compound of thisinvention is a phosphatidylethanolamine, a phosphatidylserine, aphosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or aphosphatidylglycerol. In another embodiment, said PL comprises theresidue of palmitic acid, myristic acid, myristoleic acid, palmitoleicacid, oleic acid, linoleic acid, linolenic acid, arachidonic acid,eicosapentaenoic acid, erucic acid or docosahexaenoic acid. In anotherembodiment, said PL is dimyristoyl phosphatidylethanolamine. In anotherembodiment, said PL is dipalmitoyl phosphatidylethanolamine.

In another embodiment, the polydispersity of said GAG is from about 1 to1.75. In another embodiment, the polydispersity of said GAG is fromabout 1.25 to 1.5.

In one embodiment, the lipid-polymer conjugate of this inventioncomprises a GAG wherein the average molecular weight of said GAG isbetween 5 kd to 90 kd. In another embodiment, the average molecularweight of said GAG is between 5 kD to 60 kD. In another embodiment, theaverage molecular weight of said GAG is between 5 kD to 40 kD.

In another embodiment, the average molecular weight of said GAG isbetween 5 kD to 15 kD. In another embodiment, the average molecularweight of said GAG is between 5 kD to 20 kD.

In one embodiment, low molecular weight GAG, such as sodium hyaluronateis prepared by acid hydrolysis of sodium hyaluronate as described inExample 9. In another embodiment, said acid hydrolysis compriseshydrochloric acid. In another embodiment, said acid hydrolysis comprisessulfuric acid. In another embodiment, said acid hydrolysis comprisestrifluoroacetic acid. In another embodiment, said acid hydrolysiscomprises hydrobromic acid. In another embodiment, said acid hydrolysiscomprises acetic acid. In another embodiment, the concentration of theacid in said acid hydrolysis is from about 0.1 to 12 molar. In anotherembodiment, the concentration of the acid in said acid hydrolysis isfrom about 1 to 6 molar. In another embodiment, the concentration of theacid in said acid hydrolysis is from about 6 to 12 molar. In anotherembodiment, said acid hydrolysis is carried out at a temperature between25 degrees Celsius to 100 degrees Celsius. In another embodiment, saidacid hydrolysis is carried out at a temperature between 25 degreesCelsius to 50 degrees Celsius. In another embodiment, said acidhydrolysis is carried out at a temperature between 50 degrees Celsius to100 degrees Celsius.

In one embodiment the molecular weight of hyaluronic acid andderivatives is determined by size exclusion chromatography andmultiangle light scattering (SEC-MALS) as described in Example 10. Thechromatogram and distribution diagram are stated in FIG. 19 and FIG. 20whereas the red line pertains to light scattering signal and the blueline to refractive index signal. FIG. 21 illustrates the UV spectrum.

Light scattering measurements can provide an absolute measurement ofmolar mass when used in series with a concentration sensitive detectorsuch as a refractive index detector and if the value of dn/dc(differential refractive index increment) is known. In essence, lightscattering measurements automatically provide a column calibration curvefor every sample, obviating time-consuming, conformation dependentcalibration procedure.

In one embodiment, the hyaluronan samples for SEC-MALS molecular weightdetermination are prepared by dissolving of a weighted amount of samplein a phosphate buffer. In another embodiment, the hyaluronan samples forSEC-MALS molecular weight determination are prepared by dissolving of aweighted amount of sample in an acetate buffer. In another embodiment,the hyaluronan samples for SEC-MALS molecular weight determination areprepared by dissolving of a weighted amount of sample in a tris buffer.In another embodiment, the hyaluronan samples for SEC-MALS molecularweight determination are prepared by dissolving of a weighted amount ofsample in a MES buffer.

In another embodiment, this invention provides a pharmaceuticalcomposition comprising a lipid-polymer conjugate comprising aglycosaminoglycan (GAG) conjugated to a phospholipid (PL) wherein saidconjugate is prepared by reacting said GAG with said PL in a mass_(PL)to mass_(GAG) ratio from about 0.25:15 to about 5:15, respectively. Inanother embodiment, the average molecular weight of said GAG is between5 kD to 90 kD. In another embodiment, the average molecular weight ofsaid GAG is between 5 kD to 20 kD. In another embodiment, the averagemolecular weight of said GAG is greater than 10 kD.

In one embodiment, this invention provides a lipid-polymer conjugaterepresented by the structure of the general formula (A):

-   -   wherein    -   L is a lipid or a phospholipid;    -   Z is either nothing, ethanolamine, serine, inositol, choline,        phosphate, or glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between L, Z, Y and X is either an amide or an        enteric bond;    -   wherein the molecular weight of said glycosaminoglycan is        between 5 kD and 20 kD.

In one embodiment L is a lipid. In another embodiment L is aphospholipid. In another embodiment, L is a phosphatidylethanolamine, aphosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, aphosphatidic acid or a phosphatidylglycerol. In another embodiment, Lcomprises the residue of palmitic acid, myristic acid, myristoleic acid,palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidonicacid, eicosapentaenoic acid, erucic acid or docosahexaenoic acid. Inanother embodiment, L is dimyristoyl phosphatidylethanolamine. Inanother embodiment, said L is dipalmitoyl phosphatidylethanolamine.

In another embodiment, X is hyaluronic acid, heparin, heparan sulfate,chondroitin, chondroitin sulfate, dermatan sulfate or keratan sulfate.In another embodiment, X is hyaluronic acid. In another embodiment, X isheparin. In another embodiment, X is chondroitin. In another embodiment,X is chondroitin sulfate. In another embodiment, X is dermatan sulfate,in another embodiment, X is keratan sulfate.

In another embodiment, said chondroitin sulfate ischondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof. Inanother embodiment, said dermatan sulfate is dermatan-6-sulfate,dermatan-4-sulfate or a derivative thereof.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (I):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan (GAG); and    -   n is a number from 1 to 70;    -   wherein if Y is nothing the phosphatidylserine is directly        linked to X via an amide bond and if Y is a spacer, said spacer        is directly linked to X via an amide or an esteric bond and to        said phosphatidylethanolamine via an amide bond.

In another embodiment, the molecular weight of said GAG is between 5 to20 kD.

Examples of phosphatidylethanolamine (PE) moieties are analogues of thephospholipid in which the chain length of the two fatty acid groupsattached to the glycerol backbone of the phospholipid varies from 2-30carbon atoms length, and in which these fatty acids chains containsaturated and/or unsaturated carbon atoms. In lieu of fatty acid chains,alkyl chains attached directly or via an ether linkage to the glycerolbackbone of the phospholipid are included as analogues of PE. In oneembodiment, the PE moiety is dipalmitoyl-phosphatidyl-ethanolamine. Inanother embodiment, the PE moiety isdimyristoyl-phosphatidyl-ethanolamine.

Phosphatidyl-ethanolamine and its analogues may be from various sources,including natural, synthetic, and semisynthetic derivatives and theirisomers.

Phospholipids which can be employed in lieu of the PE moiety areN-methyl-PE derivatives and their analogues, linked through the aminogroup of the N-methyl-PE by a covalent bond; N,N-dimethyl-PE derivativesand their analogues linked through the amino group of theN,N-dimethyl-PE by a covalent bond, phosphatidylserine (PS) and itsanalogues, such as palmitoyl-stearoyl-PS, natural PS from varioussources, semisynthetic PSs, synthetic, natural and artifactual PSs andtheir isomers. Other phospholipids useful as conjugated moieties in thisinvention are phosphatidylcholine (PC), phosphatidylinositol (PI),phosphatidic acid and phosphoatidylglycerol (PG), as well as derivativesthereof comprising either phospholipids, lysophospholipids, phosphatidicacid, sphingomyelins, lysosphingomyelins, ceramide, and sphingosine.

For PE-conjugates and PS-conjugates, the phospholipid is linked to theconjugated monomer or polymer moiety through the nitrogen atom of thephospholipid polar head group, either directly or via a spacer group.For PC, PI, and PG conjugates, the phospholipid is linked to theconjugated monomer or polymer moiety through either the nitrogen or oneof the oxygen atoms of the polar head group, either directly or via aspacer group.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (II):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;        -   wherein if Y is nothing the phosphatidylserine is directly            linked to X via an amide bond and if Y is a spacer, said            spacer is directly linked to X via an amide or an esteric            bond and to said phosphatidylethanolamine via an amide bond.

In one embodiment, the phosphatidylserine may be bonded to Y, or to X ifY is nothing, via the COO″ moiety of the phosphatidylserine.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (III):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, inositol, choline, or glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the phosphatidyl, Z, Y and X is either        an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (IV):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;        -   wherein any bond between the phospholipid, Z, Y and X is            either an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (V):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the phospholipid, Z, Y and X is either        an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (VI):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;        -   wherein any bond between the phospholipid, Z, Y and X is            either an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (VII):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;        -   wherein any bond between the phospholipid, Z, Y and X is            either an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (VIII):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the phospholipid, Z, Y and X is either        an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the of the general formula (IX):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer, or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the phospholipid, Z, Y and X is either        an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (IXa):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer, or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the phospholipid, Z, Y and X is either        an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (IXb):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer, or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the phospholipid, Z, Y and X is either        an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the of the general formula (X):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer, or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the ceramide phosphoryl, Z, Y and X is        either an amide or an esteric bond.

In another embodiment, the compound for use in the present invention isrepresented by the structure of the general formula (Xa):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer, or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the ceramide phosphoryl, Z, Y and X is        either an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the of the general formula (XI):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein if Y is nothing the sphingosyl is directly linked to X        via an amide bond and if Y is a spacer, the spacer is directly        linked to X and to the sphingosyl via an amide bond and to X via        an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the of the general formula (XII):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the ceramide, Z, Y and X is either an        amide or an esteric bond.

In another embodiment, the compound for use in the present invention isrepresented by the structure of the general formula (XIIa):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the ceramide, Z, Y and X is either an        amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (XIII):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, ethanolamine, serine, choline, inositol, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the diglyceryl, Z, Y and X is either an        amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (XIV):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, choline, ethanolamine, serine, inositol, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the glycerolipid, Z, Y and X is either        an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (XV):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, choline, ethanolamine, serine, inositol, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the glycerolipid, Z, Y and X is either        an amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (XVI):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, choline, ethanolamine, serine, inositol, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the lipid, Z, Y and X is either an        amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (XVII):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, choline, ethanolamine, serine, inositol, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the lipid, Z, Y and X is either an        amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (XVIII):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, choline, ethanolamine, serine, inositol, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the lipid, Z, Y and X is either an        amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (XIX):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, choline, ethanolamine, serine, inositol, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the lipid, Z, Y and X is either an        amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (XX):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, choline, ethanolamine, serine, inositol, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between the lipid, Z, Y and X is either an        amide or an esteric bond.

In another embodiment, said lipid-polymer conjugate is represented bythe structure of the general formula (XXI):

-   -   wherein    -   R₁ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is either hydrogen or a linear, saturated, mono-unsaturated,        or poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Z is either nothing, choline, ethanolamine, serine, inositol, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a physiologically acceptable monomer, dimer, oligomer or        polymer wherein X is a glycosaminoglycan; and n is a number from        1 to 70;    -   wherein any bond between the lipid, Z, Y and X is either an        amide or an esteric bond.    -   In another embodiment, R₁ of formulae (I), (II), (III), (IV),        (V), (VI), (VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI),        (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII),        (XIX), (XX), (XXI) and (XXII) is a residue of palmitic acid or a        residue of myristic acid.

In another embodiment, R₂ of formulae (I), (II), (III), (IV), (V), (VI),(VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa),(XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and(XXII) is a residue of palmitic acid or a residue of myristic acid.

In some embodiments, the compounds (A), (B) (III), (IV), (V), (VI),(VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa),(XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI) and(XXII) as presented hereinabove comprises a Z group. In one embodiment,Z is a nothing. In another embodiment Z is inositol. In anotherembodiment, Z is choline. In another embodiment, Z is glycerol. Inanother embodiment, Z is ethanoleamine. In another embodiment, Z isserine.

For any or all of the compounds represented by the structures of thegeneral formulae (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII),(IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV),(XVI), (XVII), (XVIII), (XIX), (XX), (XXI), and (XXII) hereinabove: Inone embodiment, X is a glycosaminoglycan. According to this aspect andin one embodiment, the glycosaminoglycan may be, inter alia, hyaluronicacid, heparin, heparan sulfate, chondroitin sulfate, keratin, keratansulfate, dermatan sulfate or a derivative thereof. In one embodiment,the chondroitin sulfate may be, inter alia, chondroitin-6-sulfate,chondroitin-4-sulfate or a derivative thereof. In another embodiment, Xis not a glycosaminoglycan. In another embodiment, X is apolysaccharide, which in one embodiment is a hetero-polysaccharide, andin another embodiment, is a homo-polysaccharide. In another embodiment,X is a polypyranose.

In another embodiment, the glycosaminoglycan is a polymer ofdisaccharide units. In another embodiment, the number of thedisaccharide units in the polymer is m. In another embodiment, m is anumber from 2-10,000. In another embodiment, m is a number from 2-500.In another embodiment, m is a number from 2-1000. In another embodiment,m is a number from 50-500. In another embodiment, m is a number from2-2000. In another embodiment, m is a number from 500-2000. In anotherembodiment, m is a number from 1000-2000. In another embodiment, m is anumber from 2000-5000. In another embodiment, m is a number from3000-7000. In another embodiment, m is a number from 5000-10,000. Inanother embodiment, a disaccharide unit of a glycosaminoglycan may bebound to one lipid or phospholipid moiety. In another embodiment, eachdisaccharide unit of the glycosaminoglycan may be bound to zero or onelipid or phospholipid moieties. In another embodiment, the lipid orphospholipid moieties are bound to the —COOH group of the disaccharideunit. In another embodiment, the bond between the lipid or phospholipidmoiety and the disaccharide unit is an amide bond.

In one embodiment, this invention provides lipid-GAG conjugate orphospholipid-GAG conjugate, and methods of use thereof, wherein saidconjugate represented by the structures of the general formulae (A),(I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (IXa), (IXb),(X), (Xa), (XI), (XII), (XIIa), (XIII), (XIV), (XV), (XVI), (XVII),(XVIII), (XIX), (XX), (XX), and (XXII). In another embodiment, theaverage molecular weight of said GAG is between 5 kD to 90 kD. Inanother embodiment, the average molecular weight of said GAG is between5 kD to 60 kD. In another embodiment, the average molecular weight ofsaid GAG is between 5 kD to 40 kD. In another embodiment, the averagemolecular weight of said GAG is between 5 kD to 15 kD. In anotherembodiment, the average molecular weight of said GAG is between 5 kD to20 kD. In another embodiment, the lipid-GAG conjugate is aphospholipid-GAG conjugate.

In one embodiment of the invention, Y is nothing. Non-limiting examplesof suitable divalent groups forming the optional bridging group (whichin one embodiment, is referred to as a spacer) Y, according toembodiments of the invention, are straight or branched chain alkylene,e.g., of 2 or more, preferably 4 to 30 carbon atoms, —CO-alkylene-CO,—NH-alkylene-NH—, —CO-alkylene-NH—, —NH-alkylene-NH, CO-alkylene-NH—, anamino acid, cycloalkylene, wherein alkylene in each instance, isstraight or branched chain and contains 2 or more, preferably 2 to 30atoms in the chain, —(—O—CH(CH₃)CH₂—)_(x)— wherein x is an integer of 1or more.

In one embodiment of the invention, the sugar rings of theglycosaminoglycan are intact. In another embodiment, intact refers toclosed. In another embodiment, intact refers to natural. In anotherembodiment, intact refers to unbroken.

In one embodiment of the invention, the structure of the lipid orphospholipid in any compound according to the invention is intact. Inanother embodiment, the natural structure of the lipid or phospholipidsin any compound according to the invention is maintained.

In one embodiment, the compounds for use in the present invention arebiodegradable.

In some embodiments, the compounds for use are as listed in Table 1below,

TABLE 1 Phospholipid Spacer Polymer (m.w.) PE None Hyaluronic acid(2-2000 kDa) Dimyristoyl-PE None Hyaluronic acid PE None Heparin(0.5-110 kDa) PE None Chondroitin sulfate A PE NoneCarboxymethylcellulose (20-500 kDa) PE Dicarboxylic acid + Polygeline(haemaccel) Diamine (4-40 kDa) PE None Hydroxyethylstarch PEDicarboxylic acid + Dextran Diamine (1-2,000 kDa) PE Carboxyl aminogroup Hyaluronic acid (5-20 kDa) PE Dicarboxyl group Hyaluronic acid(5-20 kDa) PE Dipalmitoic acid Hyaluronic acid (5-20 kDa) PE Carboxylamino group Heparin (5-20 kDa) PE Dicarboxyl group Heparin (5-20 kDa) PECarboxyl amino group Chondroitin sulfate A PE Dicarboxyl groupChondroitin sulfate A PE Carboxyl amino group Carboxymethylcellulose(5-20 kDa) PE Dicarboxyl group Carboxymethylcellulose (5-20 kDa) PE NonePolygeline (haemaccel) (5-20 kDa) PE Carboxyl amino group Polygeline(haemaccel) (5-20 kDa) PE Dicarboxyl group Polygeline (haemaccel) (5-20kDa) PE Carboxyl amino group Hydroxyethylstarch PE Dicarboxyl groupHydroxyethylstarch PE None Dextran (5-20 kDa) PE Carboxyl amino groupDextran (5-20 kDa) PE Dicarboxyl group Dextran (5-20 kDa) PE NoneChondroitin sulfates Dipalmitoyl-PE None Hyaluronic acid Dipalmitoyl-PENone Heparin Dipalmitoyl-PE None Chondroitin sulfate A Dipalmitoyl-PENone Carboxymethylcellulose Dipalmitoyl-PE None Polygeline (haemaccel)Dipalmitoyl-PE None Hydroxyethylstarch Dipalmitoyl-PE None DextranDimyristoyl-PE None Heparin Dimyristoyl-PE None Chondroitin sulfate ADimyristoyl-PE None Carboxymethylcellulose Dimyristoyl-PE NonePolygeline (haemaccel) Dimyristoyl-PE None HydroxyethylstarchDimyristoyl-PE None Dextran PS None Hyaluronic acid PS None Heparin PSNone Polygeline (haemaccel) PC None Hyaluronic acid PC None Heparin PCNone Polygeline (haemaccel) PI None Hyaluronic acid PI None Heparin PINone Polygeline (haemaccel) PG None Hyaluronic acid PG None Heparin PGNone Polygeline (haemaccel)

In one embodiment, this invention provides a lipid-polymer conjugaterepresented by the structure of the general formula (B):

-   -   wherein    -   L is a lipid or a phospholipid;    -   Z is either nothing, ethanolamine, serine, inositol, choline,        phosphate, or glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 10;    -   wherein any bond between L, Z, Y and X is either an amide or an        esteric bond.

In one embodiment, this invention provides a lipid-polymer conjugaterepresented by the structure of the general formula (XXII):

-   -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 10;    -   wherein if Y is nothing the phosphatidylethanolamine is directly        linked to X via an amide bond and if Y is a spacer, said spacer        is directly linked to X via an amide or an esteric bond and to        said phosphatidylethanolamine via an amide bond.

In one embodiment, n of formula (B) and formula (X) is 1-10, in anotherembodiment, n is 1. In another embodiment, n is 2. In anotherembodiment, n is 3. In another embodiment, n is 4. In anotherembodiment, n is 5. In another embodiment, n is 6. In anotherembodiment, n is 7. In another embodiment, n is 8. In anotherembodiment, n is 9. In another embodiment, n is 10.

In one embodiment, this invention provides a process for preparing acompound represented by the structure of the general formula (I):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 70;        -   wherein if Y is nothing the phosphatidylethanolamine is            directly linked to X via an amide bond and if Y is a spacer,            said spacer is directly linked to X via an amide or an            esteric bond and to said phosphatidylethanolamine via an            amide bond;    -   comprising reacting a phospholipid (PL) with a glycosaminoglycan        (GAG) and a coupling agent, wherein the mass_(PL) to mass_(GAG)        ratio from about 0.25:15 to about 5:15, respectively;

In one embodiment, this invention provides a process for preparing acompound represented by the structure of the general formula (I):

-   -   wherein    -   R₁ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   R₂ is a linear, saturated, mono-unsaturated, or        poly-unsaturated, alkyl chain ranging in length from 2 to 30        carbon atoms;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 70;        -   wherein if Y is nothing the phosphatidylethanolamine is            directly linked to X via an amide bond and if Y is a spacer,            said spacer is directly linked to X via an amide or an            esteric bond and to said phosphatidylethanolamine via an            amide bond;    -   comprising the steps of:    -   a. reacting a phospholipid (PL) with a glycosaminoglycan (GAG)        and a coupling agent, wherein the mass_(PL) to mass_(GAG) ratio        from about 0.25:15 to about 5:15, respectively;    -   b. filtering the reaction mixture from (a) to generate a        filtrate; and    -   c. extracting a product from a filtrate.

In another embodiment, said coupling agent is DCC, EDAC, BOP, PyBOP,HATU, TSTU or any other amide coupling agent. In another embodiment,said coupling agent is EDAC. In another embodiment, said coupling agentfurther comprises HOBT or HOAT.

In another embodiment, said filtering step comprises a 10 kD centrasettemembrane.

In another embodiment, R₁ is the residue of palmitic acid or the residueof myristic acid.

In another embodiment, R₂ is the residue of palmitic acid or the residueof myristic acid.

In another embodiment, the average molecular weight of theglycosaminoglycan is between 5 kD to 90 kD. In another embodiment, theaverage molecular weight of the glycosaminoglycan is between 5 kD to 20kD. In another embodiment, the average molecular weight of theglycosaminoglycan is between 5 kD to 10 kD. In another embodiment, theaverage molecular weight of the glycosaminoglycan is between 10 kD to 20kD. In another embodiment, the average molecular weight of theglycosaminoglycan is between 20 kD to 50 kD. In another embodiment, theaverage molecular weight of the glycosaminoglycan is between 30 kD to 60kD. In another embodiment, the average molecular weight of theglycosaminoglycan is between 40 kD to 70 kD. In another embodiment, theaverage molecular weight of the glycosaminoglycan is between 50 kD to 80kD. In another embodiment, the average molecular weight of theglycosaminoglycan is between 60 kD to 90 kD

In one embodiment, hyaluronic acid (HA) is used in solution form. Inanother embodiment, HA solution is prepared according to Example 1,

In one embodiment, the process for the preparation of fractionatedhyaluronic acid includes ultrafiltration. In another embodiment, theultrafiltration fractionation of hyaluronic acid is as described inExample 2.

In one embodiment, phosphatidylethanolamine-hyaluronic acid conjugate(HyPE) is prepared by reacting a GAG with a PL using a coupling agent.In another embodiment, HyPE is prepared according to Example 3 using theapparatus depicted in FIG. 1.

In one embodiment, fractionated HA is used in the preparation of HyPE.In another embodiment, fractionated HA is prepared according to Example3. In another embodiment, HyPE is prepared according to Example 11.

In one embodiment, a coupling reagent is used in the preparation of HyPEaccording to Example 3. In another embodiment, EDAC is used as thecoupling reagent. In another embodiment, DCC is used as the couplingagent. In another embodiment, BOP is used as the coupling agent. Inanother embodiment, PyBOP is used as the coupling agent. In anotherembodiment, HATU is used as the coupling agent. In another embodiment,TSTU is used as the coupling agent.

In one embodiment, the coupling agent used in the preparation of HyPEaccording to Example 3 comprises HOBT. In another embodiment, thecoupling agent comprises HOAT.

In one embodiment, crude HyPE is processed by an ultrafiltration step.In another embodiment, HyPE is subjected to the alkaline ultrafiltrationdescribed in Example 4.

In one embodiment, filtered HyPE is isolated by extraction. In anotherembodiment, HyPE is extracted according to the process described inExample 5. In another embodiment, said extraction comprisesdichloromethane, ethanol and methanol.

In one embodiment, this invention provides a method of treating aninflammatory disorder in a subject, said method comprising administeringto a subject suffering from an inflammatory disorder a compositioncomprising a lipid-polymer conjugate comprising a glycosaminoglycan(GAG) conjugated to a phospholipid (PL) wherein said conjugate isprepared by reacting said GAG with said PL in a mass_(PL) to mass_(GAG)ratio from about 0.25:15 to about 5:15, respectively.

In another embodiment, said mass_(PL) to mass_(GAG) ratio is about0.25:15. In another embodiment, said mass_(PL) to mass_(GAG) ratio isabout 0.5:15. In another embodiment, said mass_(PL) to mass_(GAG) ratiois about 1:15. In another embodiment, said mass_(PL) to mass_(GAG) ratiois about 2:15. In another embodiment, said mass_(PL) to mass_(GAG) ratiois about 5:15.

In another embodiment, said inflammatory disorder is rheumatoidarthritis, osteoarthritis, wound healing, dermatitis, restenosis, cysticfibrosis, multiple sclerosis or sepsis.

In one embodiment, in vitro assays are used to measure the ability ofHyPE and HyPE analogs to reduce the expression of pro-inflammatorycytokines. In another embodiment, cell-based assays are used accordingto Example 6, Example 7 and Example 8. In another embodiment, expressionof IL-6 is measured. In another embodiment, expression of TNF-α ismeasured. In another embodiment, expression of IP-10 is measured. Inanother embodiment, expression of PGE₂ is measured.

In another embodiment, said composition is administered intravenously.In another embodiment, said composition is administered topically.

In one embodiment, the present invention provides a method fordecreasing expression of proinflammatory chemokines, cytokines, or acombination thereof comprising the step of administering a compoundrepresented by the structure of the general formula (A):

-   -   wherein    -   L is a lipid or a phospholipid;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between L, Z, Y and X is either an amide or an        esteric bond to a subject with high levels of proinflammatory        chemokines, cytokines, or a combination thereof.

In one embodiment, the present invention provides a method of activatingNF-κB, IL-6, IL-8, or a combination thereof in human airway epithelialcell lines comprising the step of administering to a subject a compoundrepresented by the structure of the general formula (A):

-   -   wherein    -   L is a lipid or a phospholipid;    -   Z is either nothing, ethanolamine, serine, inositol, choline, or        glycerol;    -   Y is either nothing or a spacer group ranging in length from 2        to 30 atoms;    -   X is a glycosaminoglycan; and    -   n is a number from 1 to 70;    -   wherein any bond between L, Z, Y and X is either an amide or an        esteric bond.

Dosages and Routes of Administration

The methods of this invention can be adapted to the use of thetherapeutic compositions comprising Lipid-conjugates in admixture withconventional excipients, i.e. pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application which do not deleteriously react with theactive compounds. Suitable pharmaceutically acceptable carriers includebut are not limited to water, salt solutions, alcohols, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycols, gelatine,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, white paraffin, glycerol,alginates, hyaluronic acid, collagen, perfume oil, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds. They can also be combinedwhere desired with other active agents, e.g., vitamins, bronchodilators,steroids, anti-inflammatory compounds, gene therapy, i.e. sequenceswhich code for the wild-type cystic fibrosis transmembrane conductanceregulator (CFTR) receptor, surfactant proteins, etc., as will beunderstood by one skilled in the art.

In one embodiment, the invention provides for the administration of asalt of a compound as described herein as well. In one embodiment, thesalt is a pharmaceutically acceptable salt, which, in turn may refer tonon-toxic salts of compounds (which are generally prepared by reactingthe free acid with a suitable organic or inorganic base) and include,but are not limited to, the acetate, benzenesulfonate, benzoate,bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, camsylate,carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate,edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate,lactate, lactobionate, laurate, malate, maleate, mandlate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,oleate, oxalate, pamaote, palmitate, panthothenate, phosphate,diphospate, polygalacturonate, salicylate, stearate, subacetate,succinate, tannate, tartrate, teoclate, tosylate, triethiodide, andvalerate salts, as well as mixtures of these salts.

In one embodiment, the route of administration may be parenteral,enteral, or a combination thereof. In another embodiment, the route maybe intra-ocular, conjunctival, topical, transdermal, intradermal,subcutaneous, intraperitoneal, intravenous, intra-arterial, vaginal,rectal, intratumoral, parcanceral, transmucosal, intramuscular,intravascular, intraventricular, intracranial, inhalation, nasalaspiration (spray), sublingual, oral, aerosol or suppository or acombination thereof. In one embodiment, the dosage regimen will bedetermined by skilled clinicians, based on factors such as exact natureof the condition being treated, the severity of the condition, the ageand general physical condition of the patient, etc.

In general, the doses utilized for the above described purposes willvary, but will be in an effective amount to exert the desiredanti-disease effect. As used herein, the term “pharmaceuticallyeffective amount” refers to an amount of a compound of formula (I) whichwill produce the desired alleviation in symptoms or signs of disease ina patient. The doses utilized for any of the above-described purposeswill generally be from 1 to about 1000 milligrams per kilogram of bodyweight (mg/kg), administered one to four times per day, or by continuousIV infusion. When the compositions are dosed topically, they willgenerally be in a concentration range of from 0.1 to about 10% w/v,administered 1-4 times per day.

In one embodiment, the use of a single chemical entity with potentanti-oxidant, membrane-stabilizing, anti-proliferative, anti-chemokine,anti-migratory, and anti-inflammatory activity provides the desiredprotection for a subject with an inflammatory disorder, or in anotherembodiment, the methods of this invention provide for use of acombination of the compounds described. In another embodiment, thecompounds for use in the present invention may be provided in a singleformulation/composition, or in another embodiment, multiple formulationsmay be used. In one embodiment, the formulations for use in the presentinvention may be administered simultaneously, or in another embodiment,at different time intervals, which may vary between minutes, hours,days, weeks or months.

In one embodiment the compositions comprising the compounds for use inthe present invention may be administered via different routes, which inone embodiment, may be tailored to provide different compounds atdifferent sites, for example some compounds may be given parenterally toprovide for superior perfusion throughout the lung and lymphatic system,and in another embodiment, some formulations/compounds/compositions maybe provided via aerosol, or in another embodiment, intranasally, toprovide for higher lung mucosal concentration. is there something wrongwith this sentence? Seems like you need the word “higher” beforemucosal?

In one embodiment, the compounds for use in the invention may be usedfor acute treatment of temporary conditions, or may be administeredchronically, as needed. In one embodiment of the invention, theconcentrations of the compounds will depend on various factors,including the nature of the condition to be treated, the condition ofthe patient, the route of administration and the individual tolerabilityof the compositions.

In one embodiment, the methods of this invention provide for theadministration of the compounds in early life of the subject, or inanother embodiment, throughout the life of the subject, or in anotherembodiment, episodically, in response to severity or constancy ofsymptomatic stages, or in another embodiment. In another embodiment, thepatients to whom the lipid or PL conjugates should be administered arethose that are experiencing symptoms of disease or who are at risk ofcontracting the disease or experiencing a recurrent episode orexacerbation of the disease, or pathological conditions associated withthe same.

As used herein, the term “pharmaceutically acceptable carrier” refers toany formulation which is safe, and provides the appropriate delivery forthe desired route of administration of an effective amount of at leastone compound of the present invention. As such, all of theabove-described formulations of the present invention are herebyreferred to as “pharmaceutically acceptable carriers.” This term refersto as well the use of buffered formulations wherein the pH is maintainedat a particular desired value, ranging from pH 4.0 to pH 9.0, inaccordance with the stability of the compounds and route ofadministration.

For parenteral administration, particularly suitable are sterilesolutions, preferably oily or aqueous solutions, as well as suspensionsor emulsions. It is also possible to freeze-dry the new compounds anduse the lyophilates obtained, for example, for the preparation ofproducts for injection.

In one embodiment, implants or suppositories, can be used to administera lipid-GAG conjugate of this invention.

For application by inhalation, particularly for treatment of airwayobstruction or congestion, solutions or suspensions of the compoundsmixed and aerosolized or nebulized in the presence of the appropriatecarrier

For topical application, particularly for the treatment of skin diseasessuch as contact dermatitis or psoriasis, admixture of the compounds withconventional creams or delayed release patches is acceptable.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules. A syrup, elixir, or the likecan be used when a sweetened vehicle is employed. When indicated,suppositories or enema formulations may be the recommended route ofadministration.

Sustained or directed release compositions can be formulated, e.g.,liposomes or those wherein the active compound is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

It will be appreciated that the actual preferred amounts of activecompound in a specific case will vary according to the specific compoundbeing utilized, the particular compositions formulated, the mode ofapplication, and the particular situs and organism being treated.Dosages for a given host can be determined using conventionalconsiderations, e.g., by customary comparison of the differentialactivities of the subject compounds and of a known agent, e.g., by meansof an appropriate, conventional pharmacological protocol.

Methods of Use

In one embodiment of the invention, the methods of the present inventionmake use of a conjugate as described herein to treat a subject sufferingfrom an inflammatory disorder, reduce or delay the mortality of asubject suffering from an inflammatory disorder or ameliorate symptomsassociated with an inflammatory disorder.

In one embodiment, the compound for use in the present inventioncomprises dipalmitoyl phosphatidylethanolamine and heparin. In oneembodiment, the compound for use in the present invention comprisesdipalmitoyl phosphatidylethanolamine and chondroitin sulfate. In oneembodiment, the compound for use in the present invention comprisesdipalmitoyl phosphatidylethanolamine and hyaluronic acid. In oneembodiment, the compound for use in the present invention comprisesdipalmitoyl phosphatidylethanolamine and carboxymethylcellulose. In oneembodiment, the compound for use in the present invention comprisesdimyristoyl phosphatidylethanolamine and hyaluronic acid.

In one embodiment, the compound for use in the present invention is adipalmitoyl phosphatidylethanolamine conjugated via an amide or esterbond to a glycosaminoglycan. In one embodiment, the compound for use inthe present invention is a dipalmitoyl phosphatidylethanolamineconjugated via an amide or ester bond to a chondroitin sulfate, which ischondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof. Inanother embodiment, the compound for use in the present invention is adipalmitoyl phosphatidylethanolamine conjugated via an amide or esterbond to a heparin. In another embodiment, the compound for use in thepresent invention is a dipalmitoyl phosphatidylethanolamine conjugatedvia an amide or ester bond to a hyaluronic acid. In another embodiment,the compound for use in the present invention is a dimyristoylphosphatidylethanolamine conjugated via an amide or ester bond to ahyaluronic acid.

In one embodiment, the conjugates of this invention display a wide-rangecombination of cytoprotective pharmacological activities, which areuseful in the present invention. In one embodiment, the compounds may beuseful for their anti-inflammatory effects. Cellular elaboration ofcytokines and chemokines serve an important regulatory function inhealth; however, when a hyperactive response to stress or disease istriggered, these compounds may present in excess and damage tissue,thereby pushing the disease state toward further deterioration. In oneembodiment, the lipid compounds for use in the methods of thisinvention, possess a combination of multiple and potent pharmacologicaleffects, including inter-alia the ability to inhibit the extracellularform of the enzyme phospholipase A2.

Method of Treating CF

In one embodiment, the conjugates of this invention are useful inaffecting inflammation in a subject with an inflammatory disorder, wherethe subject is administered lipid-conjugates at pre-symptomatic stagesof the disease. A characteristic feature of inflammation in the CF lungis the persistent infiltration of massive numbers of neutrophils intothe airways. Although neutrophils help to control infection, whenpresent in great excess, they can be harmful. Major advances in theunderstanding of the inflammatory process in the CF lung have come fromthe use of bronchoscopy and bronchioalveolar lavage (BAL) to analyze theinflammatory process in patients who are relatively symptom free and/ordo not regularly produce sputum. Recent BAL studies suggest thatneutrophil-rich inflammation begins very early, even in infants withoutclinically apparent lung disease. Thus, in one embodiment, thelipid/phospholipid conjugates of the present invention may be useful intreating CF, even in presymptomatic stages of disease.

Thus, in one embodiment, the invention provides methods for treating asubject suffering from cystic fibrosis, reducing or delaying themortality of a subject suffering from cystic fibrosis or amelioratingsymptoms associated with cystic fibrosis, and thecompounds/compositions/formulations, in one embodiment, diminish orabrogate a deleterious inflammatory response in said subject, or inanother embodiment, prevent, treat, reduce the incidence of, reduce theseverity of, delay the onset of, or diminish the pathogenesis of aninfection is the CF subject. In another embodiment, the inventionprovides methods for decreasing expression of proinflammatorychemokines, cytokines, or a combination thereof, while in anotherembodiment, the invention provides methods of activating NF-κB, IL-6,IL-8, or a combination thereof in human airway epithelial cell lines.

Method of Treating Wounds

In another embodiment, provided herein a method for promoting woundhealing comprising applying or administering to a wound site to betreated in a subject an effective amount of a composition comprising anyconjugate as described herein. In another embodiment, provided herein amethod for promoting wound healing comprising applying or administeringto a wound site to be treated in a subject an effective amount of acomposition comprising any compound represented by the structure of thegeneral formula (A).

In another embodiment, promoting wound healing comprises inducing woundhealing. In another embodiment, promoting wound healing comprisesspeeding up wound healing. In another embodiment, promoting woundhealing comprises reducing the risk of viral and/or bacterial infection.In another embodiment, promoting wound healing comprises reducinginflammation in or near the wound site.

In another embodiment, the conjugates as described herein increase therate of chronic and acute wound healing. In another embodiment, theconjugates as described herein counteract mechanisms which delay orimpair wound healing. In another embodiment, the compounds as describedherein counteract exogenous factors which delay or impair wound healing.In another embodiment, the conjugates as described herein counteractendogenous factors which delay or impair wound healing. In anotherembodiment, factors include: infection, ulceration particularly throughdiabetes, circulation problems associated with vascular disease,malnutrition, stress, cancer radiotherapy and/or chemotherapy,compromise of the immune system or simply due to the normal agingprocess. In another embodiment, a method a described herein providesboth a therapeutic and a cosmetic approach that promote wound healingprocesses.

In another embodiment, wounds include, but are not limited to thefollowing: surgical wounds; bites; burns; acid and alkali burns; coldburn (frostbite), sun burn, minor cuts, major cuts, abrasions,lacerations, wounds caused by gunshot or knife injury; wounds caused bycongenital disorders; wounds following dental surgery; periodontaldisease; wounds following trauma; tumour associated wounds, which can beclassified as malignant cutaneous ulcers related to the primary tumouror metastases; ulcers, leg ulcers; foot ulcers; pressure sores andcorneal wounds.

Method of Treating Arthritis

In another embodiment of the invention, the methods of the presentinvention make use of a conjugate as described herein for treating asubject suffering from arthritis, reducing or delaying the damage to thejoints of a subject suffering from arthritis, or ameliorating symptomsassociated with arthritis. In another embodiment of the invention, themethods of the present invention make use of a formulation comprising aconjugate as described herein for treating a subject suffering fromarthritis, reducing or delaying the damage to the joints of a subjectsuffering from arthritis, or ameliorating symptoms associated witharthritis.

In another embodiment, provided herein a method of treating a subjectsuffering from joint pain, swelling within the joint, inflammationwithin the joint, or a combination thereof comprising the step ofadministering a composition comprising a conjugate of the invention tothe subject. In another embodiment, provided herein a method of treatinga subject suffering from joint pain, swelling within the joint,inflammation within the joint, or a combination thereof comprising thestep of injecting into a swelled/inflamed joint a composition comprisinga conjugate of the invention.

It is understood that one skilled in the art recognizes that the term“arthritis” refers to both rheumatoid arthritis (RA) and osteoarthritis(OA).

In another embodiment, a compound as described herein inhibits theproduction of IL-6, IL-8, TNF-alpha, NF-κB, or their combination,thereby reducing or delaying the damage to the joints of a subjectsuffering from arthritis. In another embodiment, a compound as describedherein inhibits the production of IL-6, IL-8, TNF-alpha, NF-κB, or theircombination, thereby ameliorating symptoms associated with arthritis. Inanother embodiment, methods comprising the administration of a conjugateas described herein treat a subject suffering from joint pain, swellingwithin the joint, inflammation within the joint, or a combinationthereof by inhibiting the production of IL-6, IL-8, TNF-alpha, NF-κB, ortheir combination. In another embodiment, locally administering acomposition comprising a conjugate as described herein by intra-jointinjection inhibits the production of IL-6, IL-8, TNF-alpha, NF-κB, ortheir combination within the joint's cells. In another embodiment,locally administering a composition comprising a conjugate as describedherein by intra-joint injection inhibits inflammation within the joint.

Method of Treating Other Inflammatory Disorders

It is understood by one skilled in the art that inflammatory disordersinclude, but are not limited to, disorders resulting from activation ofthe immune system. Thus, autoimmune disorders are understood to beinflammatory disorders. Such disorders include, but are not limited to,rheumatoid arthritis, osteoarthritis, wound healing, dermatitis,restenosis, cystic fibrosis, central nervous system tissue insult,multiple sclerosis, obstructive respiratory disease, Crohn's disease,cardiovascular disease, atherosclerosis, contact dermatitis, psoriasis,ARDS, or sepsis

In one embodiment, the invention provides a method of treating a subjectsuffering from obstructive respiratory disease, including, inter alia,the step of administering to a subject an effective amount of aconjugate of this invention, thereby treating the subject suffering fromobstructive respiratory disease.

In one embodiment, the invention provides a method of treating a subjectsuffering from colitis, Crohn's disease, or another form of intestinalmucosal injury, including, inter alia, the step of administering to asubject an effective amount of a conjugate of this invention, therebytreating the subject suffering from intestinal mucosal injury, includingcolitis or Crohn's disease.

In one embodiment, the invention provides a method of treating a subjectsuffering from cardiovascular disease, including, inter alia, the stepof administering to a subject an effective amount of a conjugate of thisinvention, thereby treating the subject suffering from a cardiovasculardisease.

The present invention provides a method of treating a subject sufferingfrom atherosclerosis, including, inter alia, the step of administeringto a conjugate of this invention, thereby treating the subject sufferingfrom atherosclerosis.

In one embodiment, the invention provides a method of treating a subjectsuffering from central nervous system tissue insult, including, interalia, the step of administering to a subject an effective amount of aconjugate of this invention, thereby treating the subject suffering froma central nervous system insult.

In one embodiment, the invention provides a method of treating a subjectsuffering from multiple sclerosis, including, inter alia, the step ofadministering to a subject an effective amount of conjugate of thisinvention, thereby treating the subject suffering from multiplesclerosis.

In one embodiment, the invention provides a method of treating a subjectsuffering from contact dermatitis, including, inter alia, the step ofadministering to a subject an effective amount of a conjugate of thisinvention, thereby treating the subject suffering from contactdermatitis.

In one embodiment, the invention provides a of treating a subjectsuffering from psoriasis, including, inter alia, the step ofadministering to a subject an effective amount of a conjugate of thisinvention, thereby treating the subject suffering from psoriasis.

In one embodiment, the invention provides a method of treating a subjectsuffering from sepsis, including, inter alia, the step of administeringto a subject an effective amount of a conjugate of this invention,thereby treating the subject suffering from sepsis.

In one embodiment, the invention provides a method of treating a subjectsuffering from ARDS, comprising the steps of administering to a subjectan effective amount of a conjugate of this invention, thereby treatingthe subject suffering from ARDS.

While pharmacological activity of the Lipid-conjugates described hereinmay be due in part to the nature of the lipid moiety, the multiple anddiverse combination of pharmacological properties observed for theLipid-conjugates may represent, in other embodiments, the ability of theconjugate to act essentially as several different drugs in one chemicalentity. Thus, for example, lung mucosal or lung parenchymal injury, asmay occur in CF, may be attenuated by any one or all of thepharmaceutical activities of immune suppression, anti-inflammation,anti-oxidation, suppression of nitric oxide production, or membranestabilization.

In one embodiment, the invention provides a method of “treating”inflammatory disorders or related diseases or disorders, which in oneembodiment, refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or lessen thetargeted pathologic condition or disorder as described hereinabove. Inone embodiment, treating refers to delaying the onset of symptoms,reducing the severity of symptoms, reducing the severity of an acuteepisode, reducing the number of symptoms, reducing the incidence ofdisease-related symptoms, reducing the latency of symptoms, amelioratingsymptoms, reducing secondary symptoms, reducing secondary infections,prolonging patient survival, preventing relapse to a disease, decreasethe number or frequency of relapse episodes, increasing latency betweensymptomatic episodes, increasing time to sustained progression,expediting remission, inducing remission, augmenting remission, speedingrecovery, or increasing efficacy of or decreasing resistance toalternate therapeutics.

In one embodiment, the methods are useful in treating an infection in asubject, wherein the pathogen is a virus or in another embodiment, thepathogen is a bacterium. In one embodiment, the infection is with apathogen which infects the respiratory system, such as mycobacteria,pseudomonas, cryptococcus, streptococcus, reovirus, influenza, or otherinfections known to those of skill in the art.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples and preferred specificembodiments are, therefore, to be construed as merely illustrative, andnot limitative of the remainder of the disclosure in any way whatsoever.

Example 1 Preparation of Hyaluronic Acid (HA) Solution

4 g of chlorocresol was dissolved in 4 L of deionized (DI) water (0.1%solution). HA UL 15 was dissolved in 4 L of 0.1% chlorocresol solutionwith mechanical stirring. To prevent clogging of the ultrafiltrationmembranes, the HA solution was filtered through a 100 μm filter followedby a 50 μm filter followed by a 10 μm filter, all previously disinfectedwith 10% hydrogen peroxide and washed with copious amounts of DI waterto ensure hydrogen peroxide has been removed (verified withperoxide-detecting strips).

Example 2 Ultrafiltration Fractionation of Hyaluronic Acid (HA)

HA solution of Example 1 was loaded into the Centramate system,previously disinfected with 10% hydrogen peroxide and washed withcopious amounts of DI water to ensure hydrogen peroxide has been removed(verified with peroxide-detecting strips).

By means of constant volume diafiltration with 70 kDa Omega TFFmembranes, 20 L of 0.1% chlorocresol solution, prepared as described inExample 1, was ultrafiltered, collecting the filtrate, the fraction lessthan 70 kDa, in a carboy, previously disinfected with 10% hydrogenperoxide. The pump speed and valves shall be set such that the retentateflow is ten times the filtrate flow and the feed pressure is less than40 PSI.

The 70 kDa membranes were replaced with 30 kDa membranes and theCentramate system was disinfected with 10% hydrogen peroxide.

5 L of the filtrate, the fraction less than 70 kDa, were loaded into thereservoir and by means of constant volume diafiltration, the remaining35 L in the carboys of the fraction less than 70 kDa were ultrafiltered.The reservoir volume was reduced to 2 L and an additional 10 L of DIwater was ultrafiltered to remove the chlorocresol (confirmed byappropriate GC assay). The reservoir volume was further reduced to 1 L,reducing the pump speed, if necessary, to keep the feed pressure below40 PSI. The reservoir was then emptied directly into an autoclavedlyoguard container, closed, frozen and lyophilized to yield HA UF 70/30.GPC analysis was performed to ensure that this lot of HA UF 70/30 wasconsistent with earlier batches. A bioburden assay and an appropriate GCassay for chlorocresol was performed. Karl Fischer analysis wasperformed to determine the water content of HA UF 70/30.

Example 3 HyPE Synthesis Reaction

Using the apparatus depicted in FIG. 36, 24 g of2-(N-morpholino)ethanesulfonic acid (MES) were dissolved in 125 mL of DIwater and the pH was adjusted to pH 6.4 by addition of 4 N NaOH.

2.5 g of dipalmitoylphosphatidylethanolamine (DPPE) and 25 g ofhydroxybenzotriazole (HOBT) were dissolved in 940 mL of tert-butanol and80 mL of water with stirring and heating at 45° C. in a 12 L roundbottom flask (forming a closed system with the pump and the sonicator,all of which will have been previously autoclaved and/or disinfectedwith 70% isopropanol). To this was added 850 mL of water and 115 mL ofthe MES solution. The pH of this solution was adjusted to pH 6.4 byaddition of 2.5 N NaOH. 25 g of HA UF 70/30 of Example 2 were thendissolved with stirring and heating at 45° C. 25 g of1-ethyl-3-(3-dimethylaminoethyl)carbodiimide (EDAC) were then added, thepump and the sonicator were turned on and the system was kept between 40and 50° C. for 3 hours. GPC analysis was performed to monitor theprogress of the reaction. After 3 hours the sonicator and the pump wereturned off and the solution was stirred at room temperature overnight.The following day 750 mL of acetonitrile were added to precipitate HyPE.This was allowed to stand for 30 minutes after which the supernatant wasremoved. To this was added 7.5 L of 2% Na₂CO₃, previously prepared bydissolving 150 g of Na₂CO₃ in 7.5 L in DI water. Vigorous mechanicalstirring for at least 2 hours hydrolyzed urea related byproducts. Thesolution was neutralized with 6 N HCl while the temperature was kept at20-25° C. by passing the solution through a cooled, jacketed flow cell.

Example 4 Alkaline Ultrafiltration of HyPE

2.25 kg of NaHCO₃ was dissolved in 150 L of 0.1% chlorocresol solution,prepared by dissolving 150 g of chlorocresol in 150 L of DI wate. Bymeans of valves, the closed reaction system was diverted so that thedigested, neutralized HyPE solution of Example 3 was pumped from theround bottom flask to the centrasette system. By means of constantvolume diafiltration with a 10 kDa Omega TFF membrane, 150 L of 1.5%NaHCO₃ in 0.1% chlorocresol solution was ultrafiltered, discarding thefiltrate, the fraction less than 10 kDa. The pump speed and valves wereset such that the retentate flow was ten times the filtrate flow and thefeed pressure was less than 40 PSI. GPC analysis was performed to ensurethe disappearance of urea-related peaks at ˜13.2 min and the HOBT peakat ˜17.2 min. The solution was neutralized with 6 N HCl while thetemperature was kept at ˜20-25° C. by passing the solution through acooled, jacketed flow cell.

Example 5 Extraction of HyPE

An extraction solution was made by mixing 3 L of dichloromethane, 3 L ofethanol and 2.25 L of methanol. 7.5 L of the extraction solution wasadded to a round bottom flask containing 3 L of crude HyPE solution ofExample 4. This was stirred vigorously for 15 minutes after which timeit was allowed to stand for 45 min. The lower dichloromethane layer wasremoved. By means of constant volume diafiltration the solution waswashed with 100 L of DI water to remove the methanol and ethanol. GPCanalysis was performed to ensure the disappearance of peaks at ˜14 min.The volume was reduced to 3 L and emptied directly into 2 autoclavedlyoguard containers, closed, frozen and lyophilized to yield HyPE. NMRand HPLC data for isolated HyPE are shown in FIG. 2 and FIG. 3.

Example 6 IN Vitro Stimulation of RAW 264.7 Cells

In vitro stimulation of RAW 364.7 cells was carried out according to theschematic depicted in FIG. 4. Each Test Article was prepared in DMEM (noFBS) at 10 mg/ml (all Test Article concentrations were corrected formoisture content), vortexed, heated at 50° C. for 5 minutes, sonicatedfor 5 minutes and filtered through a 0.2 micron syringe filter. 2× TestArticle working solutions of 0.6 mg/ml, 0.2 mg/ml and 0.06 mg/ml wereprepared by diluting the 10 mg/ml stock solutions in CM. A 2.55 mMsolution of dexamethasone prepared in ethanol was diluted to 2 μM (2×working solution) in CM. The vehicle control solution was CM. A 1 mg/mlsolution of LPS (made in 1×PBS) was diluted in CM to 10 μg/ml. RAW 264.7cells were grown for XX passages (subculture every 3-4 days) in CM at37° C. with 5% CO₂. 0.5 ml of cells at 1×106 cells/ml was plated in24-well tissue culture plates. Cells were allowed to adhere for 30minutes at 37° C. with 5% CO₂ prior to treatment. The appropriate TestArticle, dexamethasone or vehicle control working solutions were addedto the cells. Cells were incubated for 1 hour at 37° C. with 5% CO₂prior to LPS treatment. 110 μl of CM was added to the -LPS plates. 110μl of 10 μg/ml LPS was added to the +1 μg/ml LPS plates. The plates wereincubated for 24 hours at 37° C. with 5% CO₂.

Example 7 Supernatant Harvesting/XTT Assay

Cell culture supernatants were collected after 24 hours of LPS treatmentand stored at −30° C. until assayed. 400 μl of media was left in eachcell culture well for the XTT assay. 400 μl of media was added to acell-free culture well for use as a blank in the XTT assay. 200 μl ofactivated XTT reagent was added to each well. Plates were incubated for1 hour at 37° C. with 5% CO₂. 100 μl was removed from each well and readat 450 nm (630 nm correction) using a ThermoMax microplate reader(Molecular Devices, Sunnyvale, Calif.). XTT data relating to highmolecular weight HyPE compositions are shown in FIG. 5 and FIG. 6. XTTdata relating to low molecular weight HyPE compositions are shown inFIG. 22 and FIG. 23.

Example 8 Cytokine/Chemokine Assays

Cell culture supernatants were assayed for IL-6, TNF-α and IP-10 using aLuminexbased assay according to the manufacturer's instructions. Datawere collected using a Luminex 100 (Luminex Corporation, Austin, Tex.).Standard curves were generated using a 5-parameter logisticcurve-fitting equation weighted by 1/y (StarStation V 2.0; AppliedCytometry Systems, Sacramento, Calif.). Each sample reading wasinterpolated from the appropriate standard curve. Calculatedconcentrations were multiplied by the appropriate dilution factor whennecessary. TNF-α data relating to high molecular weight HyPEcompositions are shown in FIG. 7, FIG. 8 and FIG. 15. TNF-α datarelating to low molecular weight HyPE compositions are shown in FIG. 24,FIG. 25 and FIG. 32. IL-6 data relating to high molecular weight HyPEcompositions are shown in FIG. 9, FIG. 10 and FIG. 16. IL-6 datarelating to low molecular weight HyPE compositions are shown in FIG. 26,FIG. 27 and FIG. 33. IP-10 data relating to high molecular weight HyPEcompositions are shown in FIG. 11, FIG. 12 and FIG. 17. IP-10 datarelating to low molecular weight HyPE compositions are shown in FIG. 28,FIG. 29 and FIG. 34.

Cell culture supernatants were assayed for PGE₂ by ELISA following themanufacturer's instructions. Absorbance readings were detected using aThermoMax microplate reader (Molecular Devices). Standard curves weregenerated using a 4-parameter logistic curve fitting equation (SoftMaxPro 4.7.1; Molecular Devices). Each sample reading was interpolated fromthe appropriate standard curve. Duplicate interpolated sample valueswere averaged and standard deviations were calculated. Calculatedconcentrations were multiplied by the appropriate dilution factor. PGE₂data relating to high molecular weight HyPE compositions are shown inFIG. 13, FIG. 14 and FIG. 18. PGE₂ data relating to low molecular weightHyPE compositions are shown in FIG. 30, FIG. 31 and FIG. 35.

Example 9 Preparation of Low Molecular Weight Sodium Hyaluronate

Raw material of sodium hyaluronate (1.32 MDa) was degraded by acidichydrolysis. The sample solution was ultrafiltered immediately afterdegradation. The final product was prepared using spray dryer as in thecase of previous samples. In addition it was filtered with 0.2 μm filter(PALL) before drying to achieve microbial purity.

Example 10 SEC-MALS Determination of Molecular Weight

The chromatography system (Agilent, 1100 Series) consisted of a HPLCpump (G1310A), an automatic injector (G1313A) and the following columnsystem: PL aquagel-OH Mix and PL aquagel-OH 30 (300×7.5 mm, 8 μm;Agilent Technologies) columns connected in series and thermostated atambient temperature. Injection volume was 100 μl. Eluent (0.1 M sodiumphosphate buffer pH 7.5) was monitored using a DAWN-EOS multi-anglelaser light scattering photometer (18-angle, Wyatt TechnologiesCorporation) and a refractive index detector rEX Optilab (WyattTechnologies Corporation). Data acquisition and molecular weightcalculations were performed using the ASTRA V software, Version5.3.2.15. The flow rate of mobile phase was maintained at 0.8 ml/min.The specific refractive index increment (dn/dc) of 0,155 mg/ml was usedfor sodium hyaluronate.

The hyaluronan samples were prepared by dissolving of a weighted amountof sample in the phosphate buffer (concentration 20.0 mg/ml). Allsamples were stirred several hours. The solutions were filtered throughsyringe filter (0.2 μm, 25 mm diameter, Whatman) and analysed by HPLCsystem.

Light scattering measurements can provide an absolute measurement ofmolar mass when used in series with a concentration sensitive detectorsuch as a refractive index detector and if the value of dn/dc(differential refractive index increment) is known.

In essence, light scattering measurements automatically provide a columncalibration curve for every sample, obviating time-consuming,conformation dependent calibration procedure.

Known dn/dc and known calibration constant of refractive index detectoras calculated method were used. Differential refractive index increment(in mL/g) was determined by using the Wyatt Optilab refractometer.

The weight-average molecular weight of hyaluronan was verified bymeasurements of dextran standard.

The determined molecular weight and polydispersity value for lowmolecular weight hyaluronic acid were 7.86×103 g/mol and 1.32 Mw/Mn,respectively. The chromatogram and distribution diagram are stated inFIG. 19 and FIG. 20 whereas red line pertains to light scattering signaland blue line to refractive index signal. FIG. 21 illustrates the UVspectrum.

Example 11 Preparation of HyPE from 9.54 kD Hyaluronic Acid

MES buffer was prepared by dissolving 14.5 g of MES in 75 mL of DI-H₂Oand adjusting the pH to 6.4 with 4N NaOH. Using an apparatus similar tothat depicted in FIG. 1, 10.0 g of HOBT was dissolved in 225 mL ofDI-H₂O, 60 mL MES buffer, 12 mL of tert-butanol. The pH was adjusted to6.4 with 4N NaOH. 15.1 g of HA was dissolved in 350 mL of DI-H₂O. 1.25 gor DPPE was dissolved in 440 mL of tert-butanol and 90 mL DI-H₂O withheating to 55 deg C. The solutions of HA and HOBT were warmed to 35 degC. and mixed. The DPPE solution, at 50 deg C. was then added to afford aclear solution. This was allowed to cool to 43 deg C., when it was addedto the flask and circulated through the sonoreactor system (FIG. 36).Some component of the reaction mixture came out of solution and it wasnecessary to heat the reaction mixture to 49 deg C. with sonication toform a clear solution. 12.5 g of EDAC was added as a powder to thereaction mixture at a temperature of 45 deg C. Sonication began with apower of 180 watts. The reaction was monitored by GPC as shown in FIGS.37-38 and because the extent of agglomeration, as observed by the ratioof the area of the first peak to that of the second continued toincrease, the reaction was allowed to continue beyond the normal 3 h andwas continued the next day. The sonication was turned off and thereaction mixture was filtered through a 0.45 μm filter to remove a smallamount of rubber debris apparently from the stator. The solution (1200mL) was extracted with 600 mL DCM and 600 mL MeOH. The resultingemulsion quickly resolved and the aqueous layer was extracted again with500 mL DCM and 500 mL EtOH. Finally, the aqueous layer was extractedwith 250 mL DCM and 250 mL EtOH and left over the weekend. Residual DCMwas removed by rotovaporation at 35 deg C. and 200 Torr. The solutionwas then transferred to a previously cleaned centrasette ultrafiltrationsystem with a 10 kDa membrane and by constant volume diafiltration waswashed with 5 L of 1.5% NaHCO₃ to remove residual organic solvents. ThepH was then increased by slow addition of 2% Na₂CO₃ to pH 9.2. Thesolution was stirred for 1 hour at room temperature. After furtherwashing with 30 L of 1.5% NaHCO₃ the peat at ˜12.5 min had disappearedand the solution was washed with 30 L of DI-H₂O until pH 7. To removeany digestion/ultrafiltration byproducts, such as free palmitic acid,the solution was then extracted again with 1 L DCM, 1 L MeOH and 0.75 LEtOH. The aqueous layer was extracted again with 400 mL DCM and 50 mLEtOH and finally a third time with 400 mL DCM and 50 mL EtOH. ResidualDCM was removed by rotovaporation at 30 deg C. and 200 Torr. By constantvolume diafiltration residual MeOH and EtOH were removed by washing with15 L DI-H₂O. The solution was concentrated to 1 L and filtered through a0.2 μm filter into a lyoguard container and placed in the lypholizer. Itwas frozen by lowering the shelf temperature to −70 deg C. When frozen,vacuum was applied (14 mT) and the shelf temperature was raised to 30deg C. Five days later 6.134 g of HyPE was recovered with awater-corrected weight of 5.2 g which corresponds to a 42% yield basedon 12.5 g (water corrected) of HA. Total phosphorus was found to be0.28% (dry basis). By LC/MS assay, 1,456 ppm of free EDU were found andafter exposure to NaOH 12,557 ppm total EDU was found. No HOBT wasdetected and MES was less than 80 ppm. GPC of the final product is shownin FIG. 39 and NMR data are shown in FIG. 40.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

1. A lipid-polymer conjugate comprising a glycosaminoglycan (GAG)conjugated to a phospholipid (PL) wherein said conjugate is prepared byreacting said GAG with said PL in a mass_(PL) to mass_(GAG) ratio fromabout 0.25:15 to about 5:15, respectively.
 2. The lipid-polymerconjugate of claim 0, wherein said mass_(PL) to mass_(GAG) ratio isabout 0.25:15.
 3. The lipid-polymer conjugate of claim 0, wherein saidmass_(PL) to mass_(GAG) ratio is about 0.5:15.
 4. The lipid-polymerconjugate of claim 0, wherein said mass_(PL) to mass_(GAG) ratio isabout 1:15.
 5. The lipid-polymer conjugate of claim 0, wherein saidmass_(PL) to mass_(GAG) ratio is about 2:15.
 6. The lipid-polymerconjugate of claim 0, wherein said mass_(PL) to mass_(GAG) ratio isabout 5:15.
 7. The lipid-polymer conjugate of claim 0, wherein said GAGis hyaluronic acid, heparin, heparan sulfate, chondroitin, chondroitinsulfate, dermatan sulfate or keratan sulfate.
 8. The lipid-polymerconjugate of claim 0, wherein said PL is a phosphatidylethanolamine, aphosphatidylserine, a phosphatidylcholine, a phosphatidylinositol, aphosphatidic acid or a phosphatidylglycerol.
 9. The lipid-polymerconjugate of claim 0, wherein said PL comprises the residue of palmiticacid or myristic acid.
 10. The lipid-polymer conjugate of claim 0,wherein said PL is dimyristoyl phosphatidylethanolamine or dipalmitoylphosphatidylethanolamine.
 11. The lipid-polymer conjugate of claim 0,wherein the polydispersity of said GAG is from about 1 to 1.75.
 12. Thelipid-polymer conjugate of claim 11, wherein the polydispersity of saidGAG is from about 1.25 to 1.5.
 13. The lipid-polymer conjugate of claim0, wherein the average molecular weight of said GAG is between 5 KD to90 KD.
 14. The lipid-polymerconjugate of claim 13, wherein the averagemolecular weight of said GAG is between 5 KD to 20 KD. 15-23. (canceled)24. A lipid-polymer conjugate comprising a glycosaminglycan (GAG)conjugated to a phospholipid (PL) via an amide or ester linkage whereinthe molecular weight of said GAG is between 5 to 20 KD and wherein thepolydispersity of said GAG is from about 1 to 1.75.
 25. Thelipid-polymer conjugate of claim 24, wherein the polydispersity of saidGAG is from about 1.25 to 1.5.
 26. The lipid-polymer conjugate of claim24, wherein said GAG is hyaluronic acid, heparin, heparan sulfate,chondroitin, chondroitin sulfate, dermatan sulfate or keratan sulfate.27. The lipid-polymer conjugate of claim 24, wherein said PL is aphosphatidylethanolamine, a phosphatidylserine, a phosphatidylcholine, aphosphatidylinositol, a phosphatidic acid or a phosphatidylglycerol. 28.The lipid-polymer conjugate of claim 24, wherein said PL comprises theresidue of palmitic acid or myristic acid.
 29. The lipid-polymerconjugate of claim 24, wherein said PL is dimyristoylphosphatidylethanolamine or dipalmitoyl phosphatidylethanolamine.
 30. Apharmaceutical composition comprising the lipid-polymer conjugate ofclaim
 1. 31. A pharmaceutical composition comprising the lipid-polymerconjugate of claim 24.